Zika virus infection perturbs osteoblast function

Zika virus (ZIKV) infection is typically characterized by a mild self-limiting disease presenting with fever, rash, myalgia and arthralgia and severe fetal complications during pregnancy such as microcephaly, subcortical calcifications and arthrogyropsis. Virus-induced arthralgia due to perturbed osteoblast function has been described for other arboviruses. In case of ZIKV infection, the role of osteoblasts in ZIKV pathogenesis and bone related pathology remains unknown. Here, we study the effect of ZIKV infection on osteoblast differentiation, maturation and function by quantifying activity and gene expression of key biomarkers, using human bone marrow-derived mesenchymal stromal cells (MSCs, osteoblast precursors). MSCs were induced to differentiate into osteoblasts and we found that osteoblasts were highly susceptible to ZIKV infection. While infection did not cause a cytopathic effect, a significant reduction of key osteogenic markers such as ALP, RUNX2, calcium contents and increased expression of IL6 in ZIKV-infected MSCs implicated a delay in osteoblast development and maturation, as compared to uninfected controls. In conclusion, we have developed and characterized a new in vitro model to study the role of bone development in ZIKV pathogenesis, which will help to identify possible new targets for developing therapeutic and preventive measures.

. In ZIKV-infected osteoblasts derived from 2 different MSC donors, ALP activity was significantly reduced at day 11 post-infection compared to uninfected controls ( Fig. 2a,b). Additionally, ZIKV infection significantly reduced osteoblast maturation in terms of mineral content in ZIKV-infected osteoblasts compared to uninfected controls at day 18 and 21 post-infection (Fig. 2c,d).

ZIKV affects osteoblast marker genes.
To quantify the impact of ZIKV infection on the expression levels of key factors during ZIKV infection, which play an instrumental role in determining the osteoblasts phenotype, we quantified gene expression of ALP, Runt-related transcription factor 2 (RUNX2, a key transcription factor for osteoblast differentiation) and a classical inflammatory mediator interleukin 6 (IL6). A significant reduction of ALP (Fig. 3a,b) and RUNX2 expression (Fig. 3c,d) was observed in ZIKV-infected differentiating osteoblasts compared to uninfected controls (p value < 0.05) at day 7 post-infection. Interestingly, the levels of IL6 were significantly increased in infected osteoblasts ( Fig. 3e,f).

Discussion
The development of arthralgia/arthritis has been a hallmark clinical symptom in several arbovirus infections, including ZIKV, CHIKV and dengue viruses 9,15,16 . While generally not considered as severe as in CHIKV infection, arthralgia/arthritis has been reported in over 70% of ZIKV cases 6 . In this study we investigated the susceptibility of MSC-derived osteoblasts to ZIKV infection and the effects of this infection on the phenotype of osteoblasts. ZIKV readily infects differentiating primary human osteoblasts and grows to high titers within 2 days post infection. These findings are in line with a recent report which confirms the susceptibility of a human osteoblast-like cell line (HOBIT) to ZIKV 11 , as well as observations from other arthritogenic arboviruses such as CHIKV 17 . However, in contrast to previous studies we did not observe CPE in ZIKV-infected primary osteoblasts. This disparity may occur in part due to the differences in in vitro models, as primary osteoblasts derived from MSCs were used in the current study compared to an osteoblast-like cell line in the previous ZIKV study and bone fragment-derived osteoblasts used in the CHIKV study. Additionally, flavivirus replication has been shown to incompletely inhibit host cell macromolecular synthesis, which may result in non-cytopathic persistent infections [18][19][20] . In the absence of CPE, ZIKV infection resulted in a persistent infection of osteoblasts for up to 3 weeks post infection, which suggests there may be viral persistence similar to CHIKV 17 . Osteoblasts play an important role in bone remodeling, which is a tightly regulated process, requiring a balance in bone resorption and bone formation. Interestingly, persistent ZIKV infection had a direct effect on the differentiation, maturation and function of primary osteoblasts. Osteoblast differentiation comprises a highly coordinated sequence of events and is regulated by the activities of several key transcription factors. The key transcription regulator RUNX2 plays a central role by modulating the commitment of osteoprogenitors and expression of major bone matrix genes by activating different pathways 21 . Following osteoblast commitment, increased levels of ALP are considered as the marker of osteogenic differentiation as it is one of the first functional enzymes observed prior to the process of mineralization. Indeed, by reducing levels of the mineralization inhibitor pyrophosphate, ALP activity is critical for the mineralization process 22 . Thus, our findings showing significantly reduced expressions of RUNX2 and ALP in persistently infected osteoblasts followed by diminished mineral depositions are of particular interest in view of ZIKV-induced alterations in the osteoblast phenotype.
In addition to bone formation biomarkers, elevated levels of IL6 comparable to that described for other arthritogenic arboviruses were also observed following ZIKV infection. This key pro-inflammatory mediator plays a pivotal role in the pathophysiology of rheumatoid arthritis, where it has been associated with stimulating neutrophil migration, osteoclast maturation and pannus proliferation 23 . During alphavirus infection, IL6 has been shown to indirectly disturb the bone homeostasis by stimulating the induction of bone resorption mediators in osteoblast cultures, resulting in bone loss and joint inflammation 7,17 . Future studies will focus on identifying key biomarkers which will allow us to predict the osteoarticular complications and disease severity following ZIKV infection.
In conclusion, these data clearly demonstrate that osteoblasts are susceptible to infection by ZIKV and that infection results in reduced differentiation and maturation of osteoblasts. The impaired function of osteoblasts can subsequently trigger an imbalance in bone homeostasis and induce bone-related disorders. These data

Virus. Zika virus Suriname ZIKVNL00013 (ZIKVAS-Sur16) was isolated from a patient in The Netherlands
(EVAg no. 011V-01621) 12 . This strain was previously shown to have a similar phenotype as the prototypic Asian lineage of ZIKV 25 . The virus stock used in this study was grown in Vero cells and passage number 3 was used for the current study. Virus titers in the supernatants were determined by endpoint titrations on Vero cells as described previously 25 . In some experiments, dengue virus (DEN 2 16681 strain) and chikungunya virus (CHIKV/IND/NL10/152) were included as controls. Briefly, tenfold serial dilution were inoculated onto a monolayer of Vero cells in a 96-wells plate (2 × 10 4 cells/well). Cytopathic effect (CPE) was used as read out and determined at 5 days post-infection (dpi), and virus titers were calculated as the 50% tissue culture infective dose (TCID50) using the Spearman-Kärber method 26 . An initial 1:10 dilution of supernatant resulted in a detection limit of 10 1.5 TCID50/ ml.

Replication kinetics of ZIKV.
MSCs were seeded three days prior to infection (day = −3). Three days post-seeding (day = 0), MSCs were stimulated into osteoblasts by adding osteogenic medium. After 6 hours of stimulation, osteoblast cultures were infected at a multiplicity of infection (moi) of 5 with ZIKVfor 1 hour at 37 °C in 5% CO 2 . The moi was based on titers determined on Vero cells. After incubation, the supernatant was removed and cells were washed three times with αMEM medium containing 10% heat-inactivated FCS followed by osteogenic medium and cells were cultured twice weekly up to three weeks depending on the experiment. Uninfected controls were cultured in parallel. To determine the ZIKV infectious titers produced, cell supernatants were collected at different time points post infection. Supernatant was stored at −80 °C until further use. Experiments were performed in triplicate with two different MSC donors.
Immunofluorescence assay. Infected cells from the replication growth kinetics assay were fixed with 4% PFA at days 4, 7, 11 and 17 post infection, permeabilized with 70% ethanol and stained using an immunofluorescence assay (IFA) as described previously 25 . Briefly, cells were incubated with mouse monoclonal antibody anti-flavivirus group antigen (MAB10216) clone D1-4G2-4-15 (Millipore, Germany) followed by staining with goat anti-mouse IgG conjugated with Alexa Fluor 488 (Life technologies, the Netherlands). After incubation, cells were mounted in ProLong ® Diamond Antifade Mountant with DAPI (Life technologies, USA). Uninfected cells and ZIKV-infected cells stained with mouse isotype IgG2a antibody (Dako, Denmark) were used as negative controls. ZIKV-infected cells were identified by using a Zeiss LSM 700 confocal laser scanning microscope fitted on an Axio observer Z1 inverted microscope (Zeiss, Breda, the Netherlands). All images were processed using Zen 2010 software (Zeiss).
Alkaline phosphatase, mineralization, and protein assays. Alkaline phosphatase (ALP) and calcium measurements were performed at different time points based on gene expression data as described previously 13,14  Quantification of mRNA expression. RNA was extracted from ZIKV-infected and uninfected cells at different time points during osteoblast differentiation using TRIzol reagent (Thermo Fisher Scientific). RNA isolation, cDNA synthesis, and PCR reactions were performed as described previously 24 . Oligonucleotide primer pairs were designed to be either on exon boundaries or spanning at least one intron (Table 1). Gene expression was corrected for the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Statistical analysis. The statistical analyses were performed using GraphPad Prism 5.01 software. All results are expressed as means with standard error of the mean (S.E.M.). Mann Whitney U test was used for the comparison between two groups (infected versus un-infected). P value ≤ 0.05 was considered significant.

Data Availability
The data sets generated during and/or analyzed during the current study are available with the corresponding author, and can be accessed on reasonable request.